Due to its advantageous physiological activities and antioxidant action, L-ascorbic acid has been used for various purposes, including those for food products and cosmetics. L-Ascorbic acid, however, has a serious disadvantageous in that it is unstable because of its reducibility and susceptible to oxidative degradation to easily lose its physiological activities. To overcome the disadvantage, the same applicant as the present invention, as one of the co-applicants of Patent Literature 1, disclosed 2-O-α-D-glucosyl-L-ascorbic acid that is composed of one molecule of D-glucose bound to the hydroxyl group at the C-2 position of L-ascorbic acid (hereinafter, abbreviated as “ascorbic acid 2-glucoside”, throughout the specification). As outstanding characteristics, ascorbic acid 2-glucoside does not exhibit reducibility, has a satisfactory stability, and exerts the physiological activities inherent to L-ascorbic acid after being hydrolyzed in living bodies into L-ascorbic acid and D-glucose by an in vivo enzyme inherently existing in the living bodies. According to the process disclosed in Patent Literature 1, ascorbic acid 2-glucoside is formed by allowing a saccharide-transferring enzyme such as cyclomaltodextrin glucanotransferase (abbreviated as “CGTase”, hereinafter) or α-glucosidase to act on a solution containing L-ascorbic acid and an α-glucosyl saccharide compound.
In Patent Literature 2, the same applicant as the present invention succeeded in crystallizing ascorbic acid 2-glucoside from a supersaturated solution of ascorbic acid 2-glucoside and disclosed crystalline ascorbic acid 2-glucoside and a particulate composition containing the same. In Non-Patent Literature 1, the same applicant as the present invention disclosed a process for producing a high ascorbic acid 2-glucoside content product on a large scale. Until now, crystalline ascorbic acid 2-glucoside has been known to exist in an anhydrous crystalline form only. For reference, Non-Patent Literatures 2 and 3 report the results on X-ray structure analysis for crystalline ascorbic acid 2-glucoside.
In Patent Literatures 3 and 4, the same applicant as the present invention further disclosed a process for producing a high ascorbic acid 2-glucoside content product, which comprises the steps of subjecting a solution with ascorbic acid 2-glucoside formed by enzymatic reactions to a column chromatography with a strong-acid cation exchange resin, and collecting a fraction rich in ascorbic acid 2-glucoside. In Patent Literature 5, the same applicant as the present invention disclosed a process for producing a high ascorbic acid 2-glucoside content product, comprising subjecting a solution containing ascorbic acid 2-glucoside formed by enzymatic reactions to electrodialysis with an anion-exchange membrane to remove impurities such as L-ascorbic acid and saccharides from the solution; and in Patent Literature 6, the same applicant as the present invention disclosed a process for producing a high ascorbic acid 2-glucoside content product, which comprises the steps of subjecting a solution with ascorbic acid 2-glucoside to an anion-exchange resin, and selectively desorbing the ingredients adsorbed on the resin to obtain a fraction rich in ascorbic acid 2-glucoside.
In addition, Patent Literatures 7 to 11 disclose a CGTase derived from a microorganism of the species Bacillus stearothermophilus, which is now classified into a microorganism of the species Geobacillus stearothermophilus; a nucleotide sequence of a gene encoding such CGTase protein; an amino acid sequence determined from the nucleotide sequence; a mutant CGTase prepared by artificially introducing a mutation into the amino acid sequence; and a process for producing saccharides using the same. Non-Patent Literatures 4 and 5 disclose the formation of ascorbic acid 2-glucoside by allowing a CGTase derived from a microorganism of the species Bacillus stearothermophilus to act on a solution containing amylaceous substance and L-ascorbic acid, and then allowing glucoamylase to act on the resulting solution to form ascorbic acid 2-glucoside.
In Patent Literature 12, the same applicant as the present invention disclosed a process for producing ascorbic acid 2-glucoside comprising allowing either an α-isomaltosyl-glucosaccharide-forming enzyme or an α-isomaltosyl-glucosaccharide-forming enzyme and CGTase to act on a solution containing L-ascorbic acid and α-glucosyl saccharide compound to form ascorbic acid 2-glucoside. Patent Literatures 13 and 14 by the same applicant as the present invention respectively disclose that an α-isomaltosyl-glucosaccharide-forming enzyme and an α-isomaltosyl-transferring enzyme form ascorbic acid 2-glucoside by catalyzing the transfer of saccharides to L-ascorbic acid.
As for the use of ascorbic acid 2-glucoside, many proposals have been made as shown in Patent Literatures 15 to 34. Due to its advantageous properties, ascorbic acid 2-glucoside has been extensively used as a food material, food additive material, cosmetic material, quasi-drug material, or pharmaceutical material for use as in conventional L-ascorbic acid and for other uses where L-ascorbic acid could not have been used because of its unstableness.
As described above, at present, ascorbic acid 2-glucoside has been known to be produced by using various saccharide-transferring enzymes from L-ascorbic acid and an amylaceous substance as materials. Among them, to the extent of the knowledge to date of the present applicant, the method comprising allowing CGTase as a saccharide-transferring enzyme to act on a solution containing either liquefied starch or dextrin and L-ascorbic acid is an industrially advantageous method because the production yield of ascorbic acid 2-glucoside is the highest. Based on the finding, the present applicant has produced a particulate composition containing anhydrous crystalline ascorbic acid 2-glucoside by a process comprising allowing CGTase to act on a solution containing either liquefied starch or dextrin and L-ascorbic acid, and has commercialized it as a material for cosmetics/quasi-drugs and for food products and food additives under the respective product names of “AA2G” (commercialzied by Hayashibara Biochemical Laboratories, Inc., Okayama, Japan) and “ASCOFRESH” (commercialized by Hayashibara Shoji, Co., Ltd., Okayama, Japan), where the all of these conventional particulate compositions containing anhydrous crystalline ascorbic acid 2-glucoside, which have been commercialzied as such a material for cosmetics/quasi drugs and for food products and food additives, are abbreviated as “quasi-drug-grade powders”, hereinafter.
Although quasi-drug-grade powders have a relatively high ascorbic acid 2-glucoside purity of 98.0% by weight or higher product specifications and have a satisfactory flowability as powders just after their productions, they have a disadvantage in that they induce caking due to their own weights and moisture absorptions when allowed to stand under relatively high temperature and humid conditions. In view of such a disadvantage, quasi-drug-grade powders have been commercialized in a product form, enclosed in a steel can with a lid after packed in a polyethylene bag by 10-kg weight each along with a desiccant, however, the present inventors' later finding revealed that quasi-drug-grade powders even in such a product form have the disadvantage that they may often cause caking and lose their useful properties as powders when stored for a relatively long period of time. When a particulate composition containing anhydrous crystalline ascorbic acid 2-glucoside for use as a cosmetic material or quasi-drug material or as a food material or food additive material is once caked, it may cause any troubles in the steps of transporting, sieving, mixing raw materials, etc., if production plants are designed under the premise that raw materials are powders retaining flowability.
A particulate composition containing anhydrous crystalline ascorbic acid 2-glucoside (product name “Ascorbic acid 2-Glucoside 999”, Code No.: AG124, commercialized by Hayashibara Biochemical Laboratories, Inc., Okayama, Japan) (hereinafter abbreviated as “a reagent-grade powder”) (see, for example, Non-Patent Literature 6), which has been commercialized as an analytical standard reagent by the same applicant as the present invention, does not cake even under the conditions that allow a quasi-drug-grade powder to cake, and still retains its properties as a powder. Similarly as in a quasi-drug-grade powder, such a reagent-grade powder is a powder prepared by allowing CGTase to act on a solution containing L-ascorbic acid and an amylaceous substance, purifying and concentrating the obtained solution containing ascorbic acid 2-glucoside to precipitate anhydrous crystalline ascorbic acid 2-glucoside, and collecting the precipitated crystals. Such a reagent-grade powder is different from a quasi-drug-grade powder in that, in addition to conventional steps, the former needs additional steps such as a recrystallization step of dissolving the once obtained crystals and then recrystallizing the crystals, and a washing step of repeatedly washing the crystals, obtained through a recrystallization step, with refined water, etc., to increase the purity of ascorbic acid 2-glucoside to a distinctly high purity of 99.9% or higher by weight. Accordingly, even in a quasi-drug-grade powder, it can be speculated to be made into a particulate composition containing anhydrous crystalline ascorbic acid 2-glucoside that substantially, more hardly cakes, by increasing the purity of ascorbic acid 2-glucoside to a level of at least 99.9% by weight.
However, as described above, to increase the purity of ascorbic acid 2-glucoside to a level of at least 99.9% by weight, a recrystallization step and a repeating washing step with refined water, etc., should be added in addition to the usual production step, resulting in disadvantages of not only an increment of times and labors required for its production but a loss of ascorbic acid 2-glucoside in the recrystallization and washing steps, as well as a reduction of the production yield and an increment of the production cost by a large margin. Because of this, it is not a realistic option to simply increase the purity of ascorbic acid 2-glucoside to a level of at least 99.9% by weight for the purpose of obtaining a particulate composition containing anhydrous crystalline ascorbic acid 2-glucoside that substantially, more hardly cakes compared to a quasi-drug-grade powder. In addition, according to the present inventors' knowledge, a reagent-grade powder has a disadvantage that it is inferior in solubility when mixed with a hydrophilic solvent, such as an aqueous 1,3-butylene glycol solution, which is frequently used in cosmetics and quasi-drugs.
Under these circumstances, the present applicant has made trial and error efforts, revealing that, in a production method of allowing CGTase to act on a solution containing either liquefied starch or dextrin and L-ascorbic acid and then allowing glucoamylase to act on the resulting solution, in the case of increasing the production yield of ascorbic acid 2-glucoside in the solution obtained by the enzymatic reactions to a level of at least 35% by weight, a powder that significantly, more hardly cakes compared to a conventional quasi-drug-grade powder can be produced through substantially the same steps as the process for producing such a conventional quasi-drug-grade powder, without dissolving and recrystallizing the once obtained anhydrous crystalline ascorbic acid 2-glucoside; and disclosed the above finding in Patent Literature 35. In the above process, however, there exists an inconvenience in that a limited, specific CGTase should be used alone or in combination with a starch-debranching enzyme such as isoamylase to increase the production yield of ascorbic acid 2-glucoside to a level of at least 35% by weight in the reaction mixture obtained by the enzymatic reactions, and such process lacks general versatility as a production method.